Establish Tet Mutant In Zebrafish By Gene Editing Technology

Background:In vertebrates,the development of hematopoietic system,including the formation and maturation of multiple kinds of blood cells,is a dynamic process that is highly ordered and complicated and also regulated by some transcription factors.HSC(hematopoietic stem cell)is the common progenitor of all kinds of blood cells.In the course hematopoiesis,a key point is the early development of HSCs.The formation,maintaining,proliferation and migration of HSC is essential for organs formation,homeostasis maintaining and wound repair.HSCs differentiate into special progenitors and precursors which give rise to mature blood cells which is precisely regulated by various mechanisms.The deficiency of the development of hematopoietic cells may lead to leukemia of different cell types such as myeloid leukemia and lymphoid leukemia.One of the common phenomenon occurring in human cancers,including leukemia,is aberrant DNA methylation which relates to gene expression regulation and genome stability.DNA methylation refers to the process in which the 5th carbon atom of cytokine in CpG island is added with a methyl and become 5-methylcytokine.DNA methylation can affect the stability of genome and regulation of gene expression.According to current studies,aberrant DNA methylation is ubiquitous in human leukemia.TET(ten-eleven translocation)protein is a kind of alpha-ketoglutarate(a-KG)and Fe2+ dependent dioxygenase.TET proteins can turn 5-methylcytokine into 5-hydroxymethylcytokine which is an essential part of DNA de-methylation.The mutations of TET family could result in various tumors,especially leukemias.In human,TET family has 3 members,TET1,TET2 and TET3.TET1 was found in a patient with leukemia caused by t(10;11)(q22;q23)translocation and was identified as a catalyzing enzyme for the hydroxylation of 5-mC.Further studies have revealed that all of the 3 members of TET family possess this capability.Deep research into the molecular mechanisms of TET family proteins' functions may help us in better understand and explain the pathogenesis and development of leukemias.Method:We knock out tetl,tet2 and tet3 genes in AB wild-type zebrafish by TALEN and CRISPR-Cas9 and screen the adults for mutants after which we detect several blood phenotypes in mutants and compared them with wild-type control to see if the mutation of tet genes is related to hematopoiesis.Results:We establishe zebrafish mutants of tet2 and tet3 and analyzed hematopoiesis-related phenotypes in the early development of embryos to find no significance between mutants and wild-type zebrafish.Conclusion:We successfully established tet2 and tet3 mutants in zebrafish,providing several favourable animal models to further study the function of tet family in hematological disease.